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Abstract:

The present invention concerns methods for inducing an immune response to
an antigen in a patient for treating human disease by administering an
immunogenic composition wherein said patient is selected in a patient
population of interest. The present invention further concerns methods
for determining whether a subject is or is not susceptible to developing
a prophylactic or therapeutic immune response after such treatment.

Claims:

1-11. (canceled)

12. A method of inducing an immune response in a cancer patient
comprising: obtaining results from a measurement of the level of
activated NK cells in a peripheral blood sample from the patient, wherein
the level of activated NK cells is measured by determining the level of
CD16, CD56 and CD69 cell surface antigens expressed by peripheral blood
lymphocytes in the sample; and administering a composition comprising at
least one recombinant viral vector expressing at least one heterologous
nucleotide sequence to the patient if the level of activated NK cells in
the sample is measured to be less than about 5%.

13. The method of claim 12, wherein the level of activated NK cells is
measured by flow cytometry.

14. The method of claim 12, wherein the level of activated NK cells is
measured using antibodies specific for CD16, CD56 and CD69 cell surface
antigens.

26. The method of claim 12, wherein the level of activated NK cells is
measured using antibodies specific for CD16, CD56, CD69, and CD3 cell
surface antigens expressed by the peripheral blood lymphocytes in the
sample.

27. A method of inducing an immune response in a cancer patient
comprising: obtaining results from a measurement of the level of
activated NK cells in a peripheral blood sample from the patient, wherein
the level of activated NK cells is measured by determining the level of
CD16, CD56 and CD69 cell surface antigens expressed by peripheral blood
lymphocytes in the sample; and administering a composition comprising at
least one recombinant viral vector expressing at least one heterologous
nucleotide sequence to the patient if the level of activated NK cells in
the sample is measured to be less than about 3.5%.

32. The method of claim 27, wherein said patient is further treated with
a chemotherapeutic agent.

Description:

[0001] The present invention relates to the field of immunology and, in
particular, to immunotherapy of a patient against diseases caused for
example by infection or cancers. More particularly, the invention relates
to methods for predicting whether a patient is or is not susceptible to
developing a prophylactic or therapeutic immune response after such
immunotherapy. The present invention relates to methods and compositions
for improving the survival rate of patients to be treated by an
immunogenic composition, in particular a vaccine.

[0002] Traditional vaccination techniques involving the introduction into
an animal system of an antigen (e.g. peptides, proteins) which can induce
an immune response, and thereby protect said animal against infection for
example, have been known for many years. These techniques have further
included the development of both live and inactivated vaccines. Live
vaccines are typically attenuated non-pathogenic versions of an
infectious agent that are capable of priming an immune response directed
against a pathogenic version of the infectious agent.

[0003] Numerous research groups have also investigated the use of vaccines
as a potential therapeutic modality for various cancer types. This
specific type of vaccine strategy is generally referred to as
immunotherapy.

[0004] In recent years there have been advances in the development of
recombinant vaccines, especially recombinant live vaccines, in which
foreign antigens of interest are encoded and expressed from a vector.
Among them, vectors based on recombinant viruses have shown great promise
and play an important role in the development of new vaccines. Many
viruses have been investigated for their ability to express proteins from
foreign pathogens or tumoral tissue, and to induce specific immunological
responses against these antigens in vivo. Generally, these gene-based
vaccines can stimulate potent humoral and cellular immune responses and
viral vectors may be an effective strategy for both the delivery of
antigen-encoding genes and the facilitation and enhancement of antigen
presentation. In order to be utilized as a vaccine carrier, the ideal
viral vector should be safe and enable efficient presentation of required
pathogen-specific antigens to the immune system. Furthermore, the vector
system must meet criteria that enable its production on a large-scale
basis. Several viral vaccine vectors have thus emerged to date, all of
them having relative advantages and limits depending on the proposed
application (for a review on recombinant viral vaccines see for example
Harrop and Carroll, 2006, Front Biosci., 11, 804-817; Yokoyama et al.,
1997, J Vet Med Sci.,59, 311-322).

[0005] Following the observation in the early 1990's that plasmid DNA
vectors could directly transfect animal cells in vivo, significant
research efforts have also been undertaken to develop vaccination
techniques based upon the use of DNA plasmids to induce immune response,
by direct introduction into animals of DNA which encodes for antigens.
Such techniques which are widely referred as DNA vaccination have now
been used to elicit protective immune responses in large number of
disease models. For a review on DNA vaccines, see Reyes-Sandoval and
Ertl, 2001 (Current Molecular Medicine, 1, 217-243).

[0006] A general problem in vaccine field however has been the
identification of a means of inducing a sufficiently strong immune
response in vaccinated individuals to protect and/or treat against
infection and disease, and thereby to extend the survival of patient
having fatal disease, for example, cancer.

[0007] Therefore there has been for example major effort in recent years,
to discover new drug compounds that act by stimulating certain key
aspects of the immune system which will serve to increase the immune
response induced by vaccines. Most of these compounds, referred as immune
response modifiers (IRMs) or adjuvants, appear to act through basic
immune system mechanisms via Toll-like receptors (TLRs) to induce various
important cytokines biosynthesis (e.g., interferons, interleukins, tumor
necrosis factor, etc. see for example Schiller et al., 2006, Exp
Dermatol., 15, 331-341). Such compounds have been shown to stimulate a
rapid release of certain dendritic cell, monocyte/macrophage-derived
cytokines and are also capable of stimulating B cells to secrete
antibodies which play an important role in the antiviral and antitumor
activities of IRM compounds.

[0008] Alternatively, vaccination strategies have been proposed, most of
them being based on a prime-boost vaccination regimen. According to these
"prime-boost" vaccination protocols, the immune system is first induced
by administering to the patient a priming composition and then boosted by
administration of a boosting second composition (see for example
EP1411974 or US20030191076).

[0009] It has been shown that functional activation and down-regulation of
natural killer (NK) cell cytotoxicity may play a major role in
reproductive outcome (Nitrivalas et al., 2001, Human Reproduction, 16,
855-861). More specifically, Thum et al, 2004, Human Reproduction, 19,
2395-2400, have evaluated the effect of the absolute count of specific
marker expression on peripheral blood natural killer (NK) cells on
implantation and miscarriage rates after Intra Vitro Fecondation (IVF)
treatment. The authors have determined that an increase in the absolute
count of activated NK cells in the peripheral blood is associated with a
reduced rate of embryo implantation in IVF treatment. Furthermore, women
with high peripheral blood NK cell absolute count, who are able to
achieve pregnancy, have a significant higher miscarriage rate.

[0010] The Applicant has now identified new tool and vaccination strategy.
According to a first embodiment, the present Invention relates to a
method for treating a patient for human disease by administering an
immunogenic composition comprising at least one antigen wherein said
patient is selected in a patient population composed of patients that
have low levels of activated NK cells.

[0011] The present Invention thus relates to a method for treating a
patient for human disease by administering an immunogenic composition
comprising at least one antigen, said method comprising the following
steps:

[0012] selection of one patient in a patient population composed
of patients that have low levels of activated NK cells,

[0013]
administering to said selected patient the said immunogenic composition.

[0014] According to another embodiment, the present Invention relates to a
method for inducing an immune response (i.e. the raised immune response)
in a patient for treating human disease by administering an immunogenic
composition wherein said patient is selected in a patient population
composed of patients that have low levels of activated NK cells.

[0015] According to another embodiment, the present Invention relates to a
method for inducing an immune response to at least one antigen (i.e. the
raised immune response) in a patient for treating human disease by
administering an immunogenic composition wherein said patient is selected
in a patient population composed of patients that have low levels of
activated NK cells.

[0016] According to another embodiment, the present Invention relates to a
method for inducing an immune response (i.e. the raised immune response)
in a patient for treating human disease by administering an immunogenic
composition wherein said patient is selected in a patient population
composed of patients that have low levels of activated NK cells and
wherein said raised immune response is innate immune response. The innate
immune response is body's initial immune defense against pathogens and is
elicited by a variety of cells including antigen-presenting cells or
"APCs". These cells express surface and cytoplasmic receptors that
recognize molecules of foreign origin (e.g., bacterial and viral nucleic
acids, proteins, carbohydrates). Upon detecting these signals, the
dendritic cells and macrophage elicit a defensive response that includes
the release of cytokines (including interferons, TNF-α, and IL-12)
and chemokines that attract cells such as immature dendritic cells,
macrophage, NK cells, and granulocytes, to the site of challenge. The
innate immune response thus confers non-specific protection while the
body is generating the adaptive response.

[0017] The present Invention thus relates to a method for inducing an
immune response (i.e. the raised immune response) in a patient for
treating human disease by administering an immunogenic composition, said
method comprising the following steps:

[0018] selection of one patient
in a patient population composed of patients that have low levels of
activated NK cells,

[0019] administering to said selected patient the
said immunogenic composition.

[0020] According to another embodiment, the present Invention relates to a
method for inducing an immune response to at least one antigen (i.e. the
raised immune response) in a patient for treating human disease by
administering an immunogenic composition, said method comprising the
following steps:

[0021] selection of one patient in a patient
population composed of patients that have low levels of activated NK
cells,

[0022] administering to said selected patient the said immunogenic
composition.

[0023] According to another embodiment, the present Invention relates to a
method for inducing an immune response (i.e. the raised immune response)
in a patient for treating human disease by administering an immunogenic
composition wherein said raised immune response is innate immune
response, said method comprising the following steps:

[0024] selection
of patient in a patient population composed of patients that have low
levels of activated NK cells,

[0025] administering to said selected
patients the said immunogenic composition.

[0026] According to another embodiment, the present Invention relates to a
method for inducing an immune response (i.e. the raised immune response)
in a patient for treating human disease by administering an immunogenic
composition, said method comprising the following steps:

[0027]
measuring in the patient the levels of activated NK cells, and

[0028]
administering to the patient the said immunogenic composition if said
patient has low levels of activated NK cells.

[0029] According to another embodiment, the present Invention relates to a
method for inducing an immune response to at least one antigen (i.e. the
raised immune response) in a patient for treating human disease by
administering an immunogenic composition, said method comprising the
following steps:

[0030] measuring in the patient the levels of
activated NK cells, and

[0031] administering to the patient the said
immunogenic composition if said patient has low levels of activated NK
cells.

[0032] According to another embodiment, the present Invention relates to a
method for inducing an immune response (i.e. the raised immune response)
in a patient for treating human disease by administering an immunogenic
composition wherein said raised immune response is innate immune
response, said method comprising the following steps:

[0033] measuring
in the patient the levels of activated NK cells, and

[0034] administering
to the patient the said immunogenic composition if said patient has low
levels of activated NK cells.

[0035] According to another embodiment, the present Invention relates to a
method for predicting whether a patient is or is not susceptible to
developing prophylactic or therapeutic immune response by administration
of an immunogenic composition, said method comprising the steps of:

[0036] obtaining a blood sample from the patient; and

[0037] measuring
levels of activated NK cells, wherein low levels of activated NK cells
indicates that the patient is predicted to have an increased
susceptibility to develop a prophylactic or therapeutic immune response.

[0038] According to another embodiment, the present Invention relates to a
method for selecting a patient susceptible to developing prophylactic or
therapeutic immune response by administration of an immunogenic
composition, said method comprising the steps of:

[0039] obtaining a
blood sample from the patient; and

[0040] measuring levels of activated
NK cells, wherein low levels of activated NK cells indicates that the
patient has an increased susceptibility to developing a prophylactic or
therapeutic immune response.

[0041] According to another embodiment, the present Invention relates to a
method for predicting whether a patient is or is not susceptible to
respond positively to a treatment comprising administration of an
immunogenic composition, said method comprising the steps of:

[0042]
obtaining a blood sample from the patient; and

[0043] measuring levels of
activated NK cells, wherein low levels of activated NK cells indicates
that the patient is predicted to have an increased susceptibility to
develop a prophylactic or therapeutic immune response.

[0044] According to another embodiment, the present Invention relates to a
method for selecting a patient susceptible to respond positively to a
treatment comprising administration of an immunogenic composition, said
method comprising the steps of:

[0045] obtaining a blood sample from
the patient; and

[0046] measuring levels of activated NK cells, wherein
low levels of activated NK cells indicates that the patient has an
increased susceptibility to developing a prophylactic or therapeutic
immune response.

[0047] According to another embodiment, the present Invention relates to
an ex-vivo method for testing whether a patient will respond
therapeutically to a method of treatment comprising administration of an
immunogenic composition, wherein the testing method comprises the steps
of:

[0050] According to another embodiment, the present Invention relates to
an ex-vivo method for testing whether a patient will respond
therapeutically to a method of treating cancer by administration of an
immunogenic composition, wherein the testing method comprises the steps
of:

[0053] As used herein throughout the entire application, the terms "a" and
"an"are used in the sense that they mean "at least one", "at least a
first", "one or more" or "a plurality" of the referenced compounds or
steps, unless the context dictates otherwise. For example, the term "a
cell" includes a plurality of cells including a mixture thereof. More
specifically, "at least one" and "one or more" means a number which is
one or greater than one, with a special preference for one, two or three.

[0054] The term "and/or" wherever used herein includes the meaning of
"and", "or" and "all or any other combination of the elements connected
by said term".

[0055] The term "about" or "approximately" as used herein means within
20%, preferably within 10%, and more preferably within 5%.

[0056] The terms "patient", "subject" refer to a vertebrate, particularly
a member of the mammalian species and includes, but is not limited to,
domestic animals, sport animals, primates including humans.

[0057] As used herein, the term "treatment" or "treating" encompasses
prophylaxis and/or therapy. Accordingly the immunogenic compositions or
methods of the present invention are not limited to therapeutic
applications and can be used in prophylaxis ones. This is covered by the
term "to developing a prophylactic or therapeutic immune response"
herein. "Prophylaxis" is not limited to preventing immediate diseases
(e.g. infectious diseases), it further encompasses prevention of long
term consequences of these infections such as cirrhosis or cancer.

[0058] An "effective amount" or a "sufficient amount" of an active
compound is an amount sufficient to effect beneficial or desired results,
including clinical results. An effective amount can be administered in
one or more administrations. A "therapeutically effective amount" is an
amount to effect beneficial clinical results, including, but not limited
to, alleviation of one or more symptoms associated with viral infection
as well as prevention of disease (e.g. prevention of one or more symptoms
of infection).

[0059] The terms "a patient selected in a patient population composed of
patients that have low levels of activated NK cells" should be understood
as meaning a patient for who level of activated NK cells has been
measured as disclosed herein, and who has low levels of activated NK
cells. When the number of patient tested is above 1, the said patients
form a patient population.

[0063] According to the Invention, the levels of activated NK cells can be
determined on total blood sample or on isolated peripheral blood
mononuclear cells (PBMC) [e.g. by Ficoll-Hypaque purification of
peripheral blood mononuclear cells (PBMC) (Bennett & Breit 1994, J Leukoc
Biol., 56(3), 236-40), or by using Sigma Accuspin® system
(Sigma-Aldrich Ltd.) according to the manufacturer's instructions, and
the like].

[0064] According to one embodiment of the Invention, the level of
activated NK cells is determined by using antibodies.

[0065] According to one specific embodiment of the Invention, said
antibodies are monoclonal antibodies.

[0066] According to one specific embodiment of the Invention, said
antibodies are tagged for example by fluorescence, radiolabel, enzyme,
biotin, or any other methods designed to render cells labeled with said
antibodies detectable. These techniques are widely used and known in the
art.

[0067] According to one preferred embodiment of the Invention, the levels
of activated NK cells is determined by using antibodies specific for
CD16, CD56 and/or CD69, preferably by antibodies specific for CD16, CD56
and CD69. Alternatively, said levels of activated NK cells are further
determined by using antibodies specific for CD3.

[0068] Thus, for example, the levels of activated NK cells is determined
by collecting peripheral blood and incubating cells with monoclonal
antibodies (e.g. with anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD56,
anti-CD69 and/or anti-CD16). Then the levels of activated NK cells is
determined with the instrument, manufactured by Instrumentation
Laboratory-Beckman Coulter, with a He-Ne laser-ray, which recognizes
wavelengths of four different fluorochromes (fluorescein isothiocyanate
FITC, phycoerythrin PE/RD1, ECD, PC5/PE).

[0069] The levels of activated NK cells can be expressed in either (i)
percent (%) of peripheral blood lymphocytes which express CD16, CD56 and
CD69 cell surface antigens, or (ii) absolute numbers of activated NK
cells per microliter of whole peripheral blood. According to one
embodiment said peripheral blood lymphocytes have been isolated from
whole blood and stored frozen until analysis.

[0070] As used herein, the terms "low levels of activated NK cells" means
either (i) levels of activated NK cells of less than about 5%,
advantageously less than about 4.5%, preferably less than about 4% and
even more preferably less than about 3.5%, and even more particularly
less than about 3.45% or (ii) fewer than about 75, preferably fewer than
about 60, and more preferably fewer than about 50 activated NK cells,
more particularly fewer than about 35 activated NK cells, preferably
those present in the mononuclear cells population, per microliter of
whole peripheral blood.

[0071] As used herein, the terms "immunogenic composition" "vaccine
composition", "vaccine" or similar terms can be used interchangeably and
mean an agent suitable for stimulating/inducing/increasing a patient's
immune system to ameliorate a current condition or to protect against or
to reduce present or future harm or infections (including viral,
bacterial, parasitic infections), e.g., reduced tumour cell proliferation
or survival, reduced pathogen replication or spread in a patient or a
detectably reduced unwanted symptom(s) associated with a condition,
extend patient survival. Said immunogenic composition can contain (i) all
or part of at least one targeted antigen and/or (ii) at least one
recombinant vector expressing in vivo all or part of at least one
heterologous nucleotide sequence, especially an heterologous nucleotide
sequence encoding all or part of at least one targeted antigen. According
to an alternate embodiment, the immunogenic composition of the Invention
comprises (iii) at least one immune response modifier, alone or in
combination with (i) and/or (ii). Examples of such immune response
modifiers (IRMs), include the CpG oligonucleotides (see U.S. Pat. No.
6,194,388; US2006094683; WO 2004039829 for example), lipopolysaccharides,
polyinosic:polycytidylic acid complexes (Kadowaki, et al., 2001, J.
Immunol. 166, 2291-2295), and polypeptides and proteins known to induce
cytokine production from dendritic cells and/or monocyte/macrophages.
Other examples of such immune response modifiers (IRMs) are small organic
molecule such as imidazoquinolinamines, imidazopyridine amines, 6,7-fused
cycloalkylimidazopyridine amines, imidazonaphthyridine amines,
oxazoloquinoline amines, thiazoloquinoline amines and 1,2-bridged
imidazoquinoline amines (see for example U.S. Pat. No. 4,689,338; U.S.
Pat. No. 5,389,640; U.S. Pat. No. 6,110,929; and U.S. Pat. No.
6,331,539).

[0072] As used herein, the term "antigen" refers to any substance,
including complex antigen (e.g. tumour cells, virus infected cells, etc .
. . ), that is capable of being the target of an immune response. An
antigen may be the target of, for example, a cell-mediated and/or humoral
immune response raised by a patient. The term "antigen" encompasses for
example all or part of viral antigens, tumour-specific or tumour-related
antigens, bacterial antigens, parasitic antigens, allergens and the like:

[0080] According to variants of the invention, the immunogenic composition
contains at least two antigens, or an heterologous nucleotide sequence
encoding at least two antigens, or at least two heterologous nucleotide
sequences encoding at least two antigens, or any combination thereof.

[0081] According to another special embodiment, said heterologous
nucleotide sequence of the present invention, encodes all or part of HPV
antigen(s) selected in the group consisting of E6 early coding region of
HPV, E7 early coding region of HPV and derivates or combination thereof.

[0082] The HPV antigen encoded by the recombinant vector according to the
invention is selected in the group consisting of an HPV E6 polypeptide,
an HPV E7 polypeptide or both an HPV E6 polypeptide and an HPV E7
polypeptide. The present invention encompasses the use of any HPV E6
polypeptide which binding to p53 is altered or at least significantly
reduced and/or the use of any HPV E7 polypeptide which binding to Rb is
altered or at least significantly reduced (Munger et al., 1989, EMBO J.
8, 4099-4105; Crook et al., 1991, Cell 67, 547-556; Heck et al., 1992,
Proc. Natl. Acad. Sci. USA 89, 4442-4446; Phelps et al., 1992, J. Virol.
66, 2148-2427). A non-oncogenic HPV-16 E6 variant which is suitable for
the purpose of the present invention is deleted of one or more amino acid
residues located from approximately position 118 to approximately
position 122 (+1 representing the first methionine residue of the native
HPV-16 E6 polypeptide), with a special preference for the complete
deletion of residues 118 to 122 (CPEEK). A non-oncogenic HPV-16 E7
variant which is suitable for the purpose of the present invention is
deleted of one or more amino acid residues located from approximately
position 21 to approximately position 26 (+1 representing the first amino
acid of the native HPV-16 E7 polypeptide, with a special preference for
the complete deletion of residues 21 to 26 (DLYCYE). According to a
preferred embodiment, the one or more HPV-16 early polypeptide(s) in use
in the invention is/are further modified so as to improve MHC class I
and/or MHC class II presentation, and/or to stimulate anti-HPV immunity.
HPV E6 and E7 polypeptides are nuclear proteins and it has been
previously shown that membrane presentation permits to improve their
therapeutic efficacy (see for example WO99/03885). Thus, it may be
advisable to modify at least one of the HPV early polypeptide(s) so as to
be anchored to the cell membrane. Membrane anchorage can be easily
achieved by incorporating in the HPV early polypeptide a
membrane-anchoring sequence and if the native polypeptide lacks it a
secretory sequence (i.e. a signal peptide). Membrane-anchoring and
secretory sequences are known in the art. Briefly, secretory sequences
are present at the N-terminus of the membrane presented or secreted
polypeptides and initiate their passage into the endoplasmic reticulum
(ER). They usually comprise 15 to 35 essentially hydrophobic amino acids
which are then removed by a specific ER-located 1endopeptidase to give
the mature polypeptide. Membrane-anchoring sequences are usually highly
hydrophobic in nature and serves to anchor the polypeptides in the cell
membrane (see for example Branden and Tooze, 1991, in Introduction to
Protein Structure p. 202-214, NY Garland).

[0083] The choice of the membrane-anchoring and secretory sequences which
can be used in the context of the present invention is vast. They may be
obtained from any membrane-anchored and/or secreted polypeptide
comprising it (e.g. cellular or viral polypeptides) such as the rabies
glycoprotein, of the HIV virus envelope glycoprotein or of the measles
virus F protein or may be synthetic. The membrane anchoring and/or
secretory sequences inserted in each of the early HPV-16 polypeptides
used according to the invention may have a common or different origin.
The preferred site of insertion of the secretory sequence is the
N-terminus downstream of the codon for initiation of translation and that
of the membrane-anchoring sequence is the C-terminus, for example
immediately upstream of the stop codon.

[0084] The HPV E6 polypeptide in use in the present invention is
preferably modified by insertion of the secretory and membrane-anchoring
signals of the measles F protein. Optionally or in combination, the HPV
E7 polypeptide in use in the present invention is preferably modified by
insertion of the secretory and membrane-anchoring signals of the rabies
glycoprotein.

[0085] The therapeutic efficacy of the recombinant vector can also be
improved by using one or more nucleic acid encoding immunopotentiator
polypeptide(s). For example, it may be advantageous to link the HPV early
polypeptide(s) to a polypeptide such as calreticulin (Cheng et al., 2001,
J. Clin. Invest. 108, 669-678), Mycobacterium tuberculosis heat shock
protein 70 (HSP70) (Chen et al., 2000, Cancer Res. 60, 1035-1042),
ubiquitin (Rodriguez et al., 1997, J. Virol. 71, 8497-8503) or the
translocation domain of a bacterial toxin such as Pseudomonas aeruginosa
exotoxin A (ETA(dIII)) (Hung et al., 2001 Cancer Res. 61, 3698-3703).

[0086] According to another and preferred embodiment, the recombinant
vector according to the invention comprises a nucleic acid encoding one
or more early polypeptide(s) as above defined, and more particularly
HPV-16 and/or HPV-18 early E6 and/or E7 polypeptides.

[0087] According to another special and preferred embodiment, said
heterologous nucleotide sequence of the present invention, encodes all or
part of MUC 1 antigen or derivates thereof.

[0088] According to another special embodiment, said heterologous
nucleotide sequence of the present invention, encodes one or more of all
or part of the followings: HCV env protein E1 or E2, core protein, NS2,
NS3, NS4a, NS4b, NS5a, NS5b, p7 or derivates thereof. According to
another special embodiment, said heterologous nucleotide sequence of the
present invention, encodes one or more fusion protein wherein the
configuration is not native in the sense that at least one of the NS
polypeptides appears in an order which is distinct from that of the
native configuration. Thus, if the fusion protein comprises a NS3
polypeptide, a NS4A polypeptide and a NS5B polypeptide, the native
configuration would be NS3-NS4A-NS5B with NS3 at the N-terminus and NS5B
at the C-terminus. In contrast, a non-native configuration can be
NS5B-NS3-NS4A, NS5B-NS4A-NS3, NS4A-NS3-NS5B, NS4A-NS5B-NS3 or
NS3-NS5B-NS4A. In particular, the fusion protein according to the
invention comprises at least one of the followings:

[0089] A NS4A
polypeptide fused directly or through a linker to the N-terminus of a NS3
polypeptide;

[0090] A NS3 polypeptide fused directly or through a linker
to the N-terminus of a NS5B polypeptide;

[0091] A NS4B polypeptide fused
directly or through a linker to the N-terminus of a NS5B polypeptide;

[0092] A NS4A polypeptide fused directly or through a linker to the
N-terminus of a NS3 polypeptide which is fused directly or through a
linker to the N-terminus of a NS4B polypeptide; and/or

[0093] A NS3
polypeptide fused directly or through a linker to the N-terminus of a
NS4B polypeptide which is fused directly or through a linker to the
N-terminus of a NS5B polypeptide.

[0094] In such specific portions of the fusion protein of the invention,
each of the NS polypeptides can be independently native or modified. For
example, the NS4A polypeptide included in the NS4A-NS3 portion can be
native whereas the NS3 polypeptide comprises at least one of the
modifications described below.

[0095] If needed, the nucleic acid molecule in use in the invention may be
optimized for providing high level expression of the targeted antigen
(e.g. HPV early polypeptide(s)) in a particular host cell or organism,
e.g. a human host cell or organism. Typically, codon optimisation is
performed by replacing one or more "native" (e.g. HPV) codon
corresponding to a codon infrequently used in the mammalian host cell by
one or more codon encoding the same amino acid which is more frequently
used. This can be achieved by conventional mutagenesis or by chemical
synthetic techniques (e.g. resulting in a synthetic nucleic acid). It is
not necessary to replace all native codons corresponding to infrequently
used codons since increased expression can be achieved even with partial
replacement. Moreover, some deviations from strict adherence to optimised
codon usage may be made to accommodate the introduction of restriction
site(s).

[0096] As used herein, the term "recombinant vector" refers to viral as
well as non viral vectors, including extrachromosomal (e.g. episome),
multicopy and integrating vectors (i.e. for being incorporated into the
host chromosomes). Particularly important in the context of the invention
are vectors for use in gene therapy (i.e. which are capable of delivering
the nucleic acid to a host organism) as well as expression vectors for
use in various expression systems. Suitable non viral vectors include
plasmids such as pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDMB (Seed,
1987, Nature 329, 840), pVAX and pgWiz (Gene Therapy System Inc; Himoudi
et al., 2002, J. Virol. 76, 12735-12746). Suitable viral vectors may be
derived from a variety of different viruses (e.g. retrovirus, adenovirus,
AAV, poxvirus, herpes virus, measle virus, foamy virus and the like). As
used herein, the term "viral vector" encompasses vector DNA/RNA as well
as viral particles generated thereof. Viral vectors can be
replication-competent, or can be genetically disabled so as to be
replication-defective or replication-impaired. The term
"replication-competent" as used herein encompasses replication-selective
and conditionally-replicative viral vectors which are engineered to
replicate better or selectively in specific host cells (e.g. tumoral
cells).

[0097] In one aspect, the recombinant vector in use in the invention is a
recombinant adenoviral vector (for a review, see "Adenoviral vectors for
gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press). It can
be derived from a variety of human or animal sources and any serotype can
be employed from the adenovirus serotypes 1 through 51. Particularly
preferred are human adenoviruses 2 (Ad2), 5 (Ad5), 6 (Ad6), 11 (Ad11), 24
(Ad24) and 35 (Ad35). Such adenovirus are available from the American
Type Culture Collection (ATCC, Rockville, Md.), and have been the patient
of numerous publications describing their sequence, organization and
methods of producing, allowing the artisan to apply them (see for example
U.S. Pat. No. 6,133,028; U.S. Pat. No. 6,110,735; WO 02/40665; WO
00/50573; EP 1016711; Vogels et al., 2003, J. Virol. 77, 8263-8271).

[0098] The adenoviral vector in use in the present invention can be
replication-competent. Numerous examples of replication-competent
adenoviral vectors are readily available to those skill in the art (see,
for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11,
2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et
al., 2000, Nature Biotechnology 18, 723-727). For example, they can be
engineered from a wild-type adenovirus genome by deletion in the E1A CR2
domain (see for example WO00/24408) and/or by replacement of the native
E1 and/or E4 promoters with tissue, tumor or cell status-specific
promoters (see for example U.S. Pat. No. 5,998,205, WO99/25860, U.S. Pat.
No. 5,698,443, WO00/46355, WO00/15820 and WO01/36650).

[0099] Alternatively, the adenoviral vector in use in the invention is
replication-defective (see for example WO94/28152; Lusky et al., 1998, J.
Virol 72, 2022-2032). Preferred replication-defective adenoviral vectors
are E1-defective (see for example U.S. Pat. No. 6,136,594 and U.S. Pat.
No. 6,013,638), with an E1 deletion extending from approximately
positions 459 to 3328 or from approximately positions 459 to 3510 (by
reference to the sequence of the human adenovirus type 5 disclosed in the
GeneBank under the accession number M 73260 and in Chroboczek et al.,
1992, Virol. 186, 280-285). The cloning capacity can further be improved
by deleting additional portion(s) of the adenoviral genome (all or part
of the non essential E3 region or of other essential E2, E4 regions).
Insertion of a nucleic acid in any location of the adenoviral vector can
be performed through homologous recombination as described in Chartier et
al. (1996, J. Virol. 70, 4805-4810). For example, the nucleic acid
encoding the HPV-16 E6 polypeptide can be inserted in replacement of the
E1 region and the nucleic acid encoding the HPV-16 E7 polypeptide in
replacement of the E3 region or vice versa.

[0100] In another and preferred aspect, the vector in use in the invention
is a poxviral vector (see for example Cox et al. in "Viruses in Human
Gene Therapy" Ed J. M. Hos, Carolina Academic Press). According to
another preferred embodiment it is selected in the group consisting of
vaccinia virus, suitable vaccinia viruses include without limitation the
Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563;
Johnson et al., 1993, Virol. 196, 381-401), the Wyeth strain and the
highly attenuated attenuated virus derived thereof including MVA (for
review see Mayr, A., et al., 1975, Infection 3, 6-14) and derivates
thereof (such as MVA vaccinia strain 575 (ECACC V00120707--U.S. Pat. No.
6,913,752), NYVAC (see WO 92/15672--Tartaglia et al., 1992, Virology,
188, 217-232). Determination of the complete sequence of the MVA genome
and comparison with the Copenhagen VV genome has allowed the precise
identification of the seven deletions (I to VII) which occurred in the
MVA genome (Antoine et al., 1998, Virology 244, 365-396), any of which
can be used to insert the antigen-encoding nucleic acid. The vector may
also be obtained from any other member of the poxviridae, in particular
fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84,
159-163); canarypox (e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev
Biol Stand., 84, 159-163); pigeonpox; swinepox and the like. By way of
example, persons skilled in the art may refer to WO 92 15672
(incorporated by reference) which describes the production of expression
vectors based on poxviruses capable of expressing such heterologous
nucleotide sequence, especially nucleotide sequence encoding antigen.

[0101] The basic technique for inserting the nucleic acid and associated
regulatory elements required for expression in a poxviral genome is
described in numerous documents accessible to the man skilled in the art
(Paul et al., 2002, Cancer gene Ther. 9, 470-477; Piccini et al., 1987,
Methods of Enzymology 153, 545-563; U.S. Pat. No. 4,769,330; U.S. Pat.
No. 4,772,848; U.S. Pat. No. 4,603,112; U.S. Pat. No. 5,100,587 and U.S.
Pat. No. 5,179,993). Usually, one proceed through homologous
recombination between overlapping sequences (i.e. desired insertion site)
present both in the viral genome and a plasmid carrying the nucleic acid
to insert.

[0102] The nucleic acid encoding the antigen of the Invention is
preferably inserted in a nonessential locus of the poxviral genome, in
order that the recombinant poxvirus remains viable and infectious.
Nonessential regions are non-coding intergenic regions or any gene for
which inactivation or deletion does not significantly impair viral
growth, replication or infection. One may also envisage insertion in an
essential viral locus provided that the defective function is supplied in
trans during production of viral particles, for example by using an
helper cell line carrying the complementing sequences corresponding to
those deleted in the poxviral genome.

[0104] When using MVA, the antigen-encoding nucleic acid can be inserted
in any one of the identified deletions I to VII as well as in the D4R
locus, but insertion in deletion II or III is preferred (Meyer et al.,
1991, J. Gen. Virol. 72, 1031-1038; Sutter et al., 1994, Vaccine 12,
1032-1040).

[0105] When using fowlpox virus, although insertion within the thymidine
kinase gene may be considered, the antigen-encoding nucleic acid is
preferably introduced in the intergenic region situated between ORFs 7
and 9 (see for example EP 314 569 and U.S. Pat. No. 5,180,675).

[0106] According to one special embodiment, said recombinant vector is a
recombinant plasmid DNA or a recombinant viral vector.

[0107] According to another special embodiment, said recombinant viral
vector is a recombinant adenoviral vector.

[0108] According to another special embodiment, said recombinant viral
vector is a recombinant vaccinia vector.

[0109] According to another special embodiment, said recombinant vaccinia
vector is a recombinant MVA vector.

[0110] Preferably, the antigen-encoding nucleic acid in use in the
invention is in a form suitable for its expression in a host cell or
organism, which means that the nucleic acid sequence encoding the antigen
are placed under the control of one or more regulatory sequences
necessary for its expression in the host cell or organism. As used
herein, the term "regulatory sequence" refers to any sequence that
allows, contributes or modulates the expression of a nucleic acid in a
given host cell, including replication, duplication, transcription,
splicing, translation, stability and/or transport of the nucleic acid or
one of its derivative (i.e. mRNA) into the host cell. It will be
appreciated by those skilled in the art that the choice of the regulatory
sequences can depend on factors such as the host cell, the vector and the
level of expression desired. The nucleic acid encoding the antigen is
operatively linked to a gene expression sequence which directs the
expression of the antigen nucleic acid within a eukaryotic cell. The gene
expression sequence is any regulatory nucleotide sequence, such as a
promoter sequence or promoter-enhancer combination, which facilitates the
efficient transcription and translation of the antigen nucleic acid to
which it is operatively linked. The gene expression sequence may, for
example, be a mammalian or viral promoter, such as a constitutive or
inducible promoter. Constitutive mammalian promoters include, but are not
limited to, the promoters for the following genes: hypoxanthine
phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,
b-actin promoter and other constitutive promoters. Exemplary viral
promoters which function constitutively in eukaryotic cells include, for
example, promoters from the cytomegalovirus (CMV), simian virus (e.g.,
SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV),
Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of
Moloney leukemia virus and other retroviruses, and the thymidine kinase
promoter of herpes simplex virus. Other constitutive promoters are known
to those of ordinary skill in the art. The promoters useful as gene
expression sequences of the invention also include inducible promoters.
Inducible promoters are expressed in the presence of an inducing agent.

[0111] For example, the metallothionein promoter is induced to promote
transcription and translation in the presence of certain metal ions.
Other inducible promoters are known to those of ordinary skill in the
art. In general, the gene expression sequence shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences involved
with the initiation of transcription and translation, respectively, such
as a TATA box, capping sequence, CAAT sequence, and the like. Especially,
such 5' non-transcribing sequences will include a promoter region which
includes a promoter sequence for transcriptional control of the operably
joined antigen nucleic acid. The gene expression sequences optionally
include enhancer sequences or upstream activator sequences as desired.
Preferred promoters for use in a poxviral vector (see below) include
without limitation vaccinia promoters 7.5K, H5R, TK, p28, p11 and K1L,
chimeric promoters between early and late poxviral promoters as well as
synthetic promoters such as those described in Chakrabarti et al. (1997,
Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological
Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158).

[0112] The promoter is of special importance and the present invention
encompasses the use of constitutive promoters which direct expression of
the nucleic acid in many types of host cells and those which direct
expression only in certain host cells or in response to specific events
or exogenous factors (e.g. by temperature, nutrient additive, hormone or
other ligand). Suitable promoters are widely described in literature and
one may cite more specifically viral promoters such as RSV, SV40, CMV and
MLP promoters. Preferred promoters for use in a poxviral vector include
without limitation vaccinia promoters 7.5K, H5R, TK, p28, p11 and K1L,
chimeric promoters between early and late poxviral promoters as well as
synthetic promoters such as those described in Chakrabarti et al. (1997,
Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological
Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158).

[0114] Alternatively, the recombinant vector in use in the present
invention can further comprise at least one nucleic acid encoding at
least one cytokine. Suitable cytokines include without limitation
interleukins (e.g. IL-2, IL-7, IL-15, IL-18, IL-21) and interferons (e.g.
IFNγ, INFα), with a special preference for interleukin IL-2.
When the recombinant vaccine of the invention comprises a
cytokine-expressing nucleic acid, said nucleic acid may be carried by the
recombinant vector encoding the one or more antigen(s) or by an
independent recombinant vector which can be of the same or a different
origin.

[0115] According to one preferred embodiment, the recombinant vector in
use in the present invention is encoding all or part of the MUC1 antigen
and at least one cytokines above listed, and preferably an interleukin,
especially IL2. Preferably the recombinant vector in use in the present
invention is an MVA encoding all or part of the MUC1 antigen and at least
one cytokines above listed, and preferably an interleukin, especially
IL2.

[0116] Infectious viral particles comprising the above-described
recombinant viral vector can be produced by routine process. An exemplary
process comprises the steps of:

[0117] a. introducing the viral vector into a suitable cell line,

[0118] b. culturing said cell line under suitable conditions so as to
allow the production of said infectious viral particle,

[0119] c. recovering the produced infectious viral particle from the
culture of said cell line, and

[0123] The infectious viral particles may be recovered from the culture
supernatant or from the cells after lysis (e.g. by chemical means,
freezing/thawing, osmotic shock, mechanic shock, sonication and the
like). The viral particles can be isolated by consecutive rounds of
plaque purification and then purified using the techniques of the art
(chromatographic methods, ultracentrifugation on caesium chloride or
sucrose gradient).

[0124] If desired, the method or use for treating a patient for human
disease human disease according to the Invention (i.e. by administering
an immunogenic composition comprising at least one antigen) can be
carried out in the selected patients in conjunction with one or more
conventional therapeutic modalities (e.g. radiation, chemotherapy and/or
surgery). The use of multiple therapeutic approaches provides the
selected patient with a broader based intervention. In one embodiment,
the method or use for treating a patient for human disease human disease
according to the Invention can be preceded or followed by a surgical
intervention. In another embodiment, it can be preceded or followed by
radiotherapy (e.g. gamma radiation). Those skilled in the art can readily
formulate appropriate radiation therapy protocols and parameters which
can be used (see for example Perez and Brady, 1992, Principles and
Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using
appropriate adaptations and modifications as will be readily apparent to
those skilled in the field). In still another embodiment, the method or
use of the invention is associated to chemotherapy with one or more drugs
(e.g. drugs which are conventionally used for treating or preventing
viral infections, virus-associated pathologic conditions, cancer, and the
like).

[0125] The present Invention thus relates to a method for improving the
treatment of a cancer patient which is undergoing chemotherapeutic
treatment with a chemotherapeutic agent, said method comprising the
following steps:

[0126] selection of patient in a patient population
composed of patients that have low levels of activated NK cells,

[0127]
administering to said selected patients an immunogenic composition
according to the Invention and a chemotherapeutic agent.

[0128] The present Invention thus relates to a method for improving the
treatment of a cancer patient which is undergoing chemotherapeutic
treatment with a chemotherapeutic agent, said method comprising the
following steps:

[0129] measuring in the patient the levels of
activated NK cells, and

[0130] administering to the patient the said
immunogenic composition if said patient has low levels of activated NK
cells according to the Invention.

[0131] According to one embodiment, the administration of said
chemotherapeutic agent is done before administration of said immunogenic
composition.

[0132] According to another embodiment, the administration of said
chemotherapeutic agent is done after administration of said immunogenic
composition.

[0133] According to another embodiment, the administration of said
chemotherapeutic agent is done concomitantly with administration of said
immunogenic composition.

[0134] According to one embodiment, said chemotherapeutic agent is
cisplatin and/or Gemcitabine, or equivalent thereof.

[0135] The present Invention further concerns a method of improving
cytotoxic effectiveness of cytotoxic drugs or radiotherapy which
comprises co-treating a patient selected in a patient population composed
of patients that have low levels of activated NK cells with an
immunogenic composition according to the Invention.

[0136] In another embodiment, the method or use of the invention is
carried out according to a prime boost therapeutic modality which
comprises sequential administration of one or more primer composition(s)
and one or more booster composition(s). Typically, the priming and the
boosting compositions use different vehicles which comprise or encode at
least an antigenic domain in common. The priming composition is initially
administered to the host organism and the boosting composition is
subsequently administered to the same host organism after a period
varying from one day to twelve months. The method of the invention may
comprise one to ten sequential administrations of the priming composition
followed by one to ten sequential administrations of the boosting
composition. Desirably, injection intervals are a matter of one week to
six months. Moreover, the priming and boosting compositions can be
administered at the same site or at alternative sites by the same route
or by different routes of administration.

[0137] According to one special embodiment, the Invention relates to a
method as above described wherein said human disease is cancer.

[0139] According to one special embodiment, the Invention relates to a
method as above described wherein said human disease is infectious
disease.

[0140] According to a preferred embodiment, said infectious disease is a
viral induced disease, such as for example disease induced by HIV, HCV,
HBV, HPV, and the like.

[0141] In a further embodiment there is provided the use of an immunogenic
composition comprising all or part of a targeted antigen for the
manufacture of a medicament for treating a patient for human disease in a
particular patient population wherein the patients of said population
have low levels of activated NK cells.

[0142] In a further embodiment there is provided the use of an immunogenic
composition for the manufacture of a medicament for inducing an immune
response (i.e. the raised immune response) in a patient for treating
human disease in a particular patient population wherein the patients of
said population have low levels of activated NK cells.

[0143] In another embodiment there is provided the use of an immunogenic
composition for the manufacture of a medicament for inducing an immune
response to at least one antigen (i.e. the raised immune response) in a
patient for treating human disease in a particular patient population
wherein the patients of said population have low levels of activated NK
cells.

[0144] In another embodiment there is provided the use of an immunogenic
composition for the manufacture of a medicament for inducing an immune
response to a targeted antigen (i.e. the raised immune response) in a
patient for treating human disease in a particular patient population
wherein the patients of said population have low levels of activated NK
cells.

[0145] In another embodiment there is provided the use of an immunogenic
composition for the manufacture of a medicament for inducing an immune
response (i.e. the raised immune response) in a patient for treating
human disease in a particular patient population wherein the patients of
said population have low levels of activated NK cells and wherein said
raised immune response is innate immune response.

[0146] According to one special embodiment, said "raised immune response"
in said patient population is directed towards a tumour-specific or
-related antigens and/or viral antigen. According to one embodiment, said
"raised immune response" in said patient population is directed towards
distinct antigens. According to one special embodiment, said "raised
immune response" in said patient population is directed towards all or
part of MUC1 antigen. According to another special embodiment, said
"raised immune response" in said patient population is T cell immune
response, and preferably CD8+ (Cytotoxic T Lymphocytes) immune response.
According to another special embodiment, said "raised immune response" in
said patient population is a non specific immune response. According to
another special embodiment, said "raised immune response" in said patient
population is a stimulation of the innate immune response.

[0147] The ability to induce or stimulate an immune response upon
administration in an animal or human organism can be evaluated either in
vitro or in vivo using a variety of assays which are standard in the art.
For a general description of techniques available to evaluate the onset
and activation of an immune response, see for example Coligan et al.
(1992 and 1994, Current Protocols in Immunology; ed J Wiley & Sons Inc,
National Institute of Health). Measurement of cellular immunity can be
performed by measurement of cytokine profiles secreted by activated
effector cells including those derived from CD4+ and CD8+ T-cells (e.g.
quantification of IL-10 or IFN gamma-producing cells by ELIspot), by
determination of the activation status of immune effector cells (e.g. T
cell proliferation assays by a classical [3H] thymidine uptake), by
assaying for antigen-specific T lymphocytes in a sensitized patient (e.g.
peptide-specific lysis in a cytotoxicity assay) or by detection of
antigen specific T cells by fluorescent MHC and/or peptide multimers
(e.g. tetramers). The ability to stimulate a humoral response may be
determined by antibody binding and/or competition in binding (see for
example Harlow, 1989, Antibodies, Cold Spring Harbor Press). The method
of the invention can also be further validated in animal models
challenged with an appropriate tumor-inducing agent (e.g. MUC1-expressing
TC1 cells) to determine anti-tumor activity, reflecting an induction or
an enhancement of an anti-antigen immune response.

[0148] Thus the present invention further concerns a method for extending
the survival of a patient treated for human disease, for example cancer,
by administering an immunogenic composition, said method comprising the
following steps:

[0149] selection of patient in a patient population
composed of patients that have low levels of activated NK cells,

[0150]
administering to said selected patients the said immunogenic composition.

[0151] The present invention further concerns a method for extending the
survival rate of a patient treated for human disease, for example cancer,
by administering an immunogenic composition, said method comprising the
following steps:

[0152] measuring in the patient the levels of
activated NK cells, and

[0153] administering to the patient the said
immunogenic composition if said patient has low levels of activated NK
cells.

[0154] The present invention further concerns a method for extending the
survival rate of a patient treated for human disease, for example cancer,
by administering an immunogenic composition and chemotherapeutic agent
(see above), said method comprising the following steps:

[0155]
measuring in the patient the levels of activated NK cells, and

[0156]
administering to the patient the said immunogenic composition and
chemotherapeutic agent if said patient has low levels of activated NK
cells.

[0157] According to another embodiment, the present Invention relates to
the use of activated NK cells as a biomarker for predicting whether a
patient is or is not susceptible to developing prophylactic or
therapeutic immune response by administration of an immunogenic
composition.

[0158] More specifically, the present Invention relates to the use of the
level of activated NK cells as a biomarker for predicting whether a
patient is or is not susceptible to developing prophylactic or
therapeutic immune response by administration of an immunogenic
composition, wherein low levels of activated NK cells indicates that the
patient is predicted to have an increased susceptibility to develop a
prophylactic or therapeutic immune response.

[0159] In other words, the present Invention relates to the use of the
level of activated NK cells as a biomarker for predicting whether a
patient is or is not susceptible to survive longer after administration
of an immunogenic composition, wherein low levels of activated NK cells
indicates that the patient is predicted to have a longer survival rate
compared to treated patients who have higher levels of activated NK
cells.

[0160] Thus the Invention further concerns the use of the level of
activated NK cells as a biomarker for predicting whether a patient which
is undergoing chemotherapeutic treatment with a chemotherapeutic agent is
or is not susceptible to developing prophylactic or therapeutic immune
response (e.g. to survive longer) after administration of an immunogenic
composition.

[0161] Thus the Invention further concerns the use of the level of
activated NK cells as a biomarker for predicting whether a patient which
is undergoing chemotherapeutic treatment with a chemotherapeutic agent is
or is not susceptible to developing prophylactic or therapeutic immune
response (e.g. to survive longer) after administration of an immunogenic
composition, wherein low levels of activated NK cells indicates that the
patient is predicted to have an increased susceptibility to develop a
prophylactic or therapeutic immune response.

[0162] The present Invention thus relates to a method for improving the
treatment of a cancer patient which is undergoing chemotherapeutic
treatment with a chemotherapeutic agent, said method comprising the
following steps:

[0163] measuring in the patient the levels of
activated NK cells, and

[0164] administering to the patient the said
immunogenic composition if said patient has low levels of activated NK
cells according to the Invention.

[0165] The present invention further concerns a method for extending the
survival of a patient treated for human disease, for example cancer, by
administering an immunogenic composition, said method comprising the
following steps:

[0166] measuring in the patient the levels of
activated NK cells, and

[0167] administering to the patient the said
immunogenic composition if said patient has low levels of activated NK
cells.

[0168] The present invention further concerns a method for extending the
survival of a patient treated for human disease, for example cancer, by
administering an immunogenic composition and chemotherapeutic agent (see
above), said method comprising the following steps:

[0169] measuring in
the patient the levels of activated NK cells, and

[0170] administering to
the patient the said immunogenic composition and chemotherapeutic agent
if said patient has low levels of activated NK cells.

[0171] The invention also provides kits (i.e. companion test) which
include parts for practicing the methods described herein and that will
be apparent from the examples provided herein. The kit of parts, or kits,
may include reagents for collecting and or measuring serum levels of
activated NK cells. Such reagents may include antibodies. The kits may
further include equipment for collecting and/or processing biological
samples. The kits are also likely to contain instructions for use,
cut-off values and/or instructions for their determination, and
instructions for interpreting the data obtained from the use of the kits.

[0172] According to one special embodiment, the said kit of parts, or
kits, may further include an immunogenic composition as above disclosed,
and/or as disclosed in the Example section below.

[0173] The invention further provides computer programs and/or algorithms
for monitoring clinical trial and activated NK cells levels, determining
whether such levels are above or below a threshold level, and/or
recommending modifications to a treatment regiment to improve a patient's
response to an immunotherapy treatment. The computer programs or
algorithms may be provided along with necessary hardware, e.g., in the
form of a kit or apparatus, which may also accept biological samples and
measure the relative levels of activated NK cells present therein. The
above-described computer programs and/or apparatus are likely to be
provided to physicians or clinical laboratories with appropriate
instructions and reagents, including antibodies.

[0174] The invention has been described in an illustrative manner, and it
is to be understood that the terminology which has been used is intended
to be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are
possible in light of the above teachings. It is therefore to be
understood that within the scope of the appended claims, the invention
may be practiced in a different way from what is specifically described
herein.

[0175] All of the above cited disclosures of patents, publications and
database entries are specifically incorporated herein by reference in
their entirety to the same extent as if each such individual patent,
publication or entry were specifically and individually indicated to be
incorporated by reference.

[0203] Blood samples were taken prior to treatment and were shipped
immediately to a central immunology lab where peripheral blood
mononuclear cells (PBMC) were isolated from the blood and stored frozen
until analysis.

[0204] PBMC were assessed for the expression of CD16, CD56 and CD69 by
flow cytometry using monoclonal antibodies specific for those antigens
(IM0814, IM2073 and IM2656, respectively, all from Beckman Coulter). The
proportion of cells expressing these antigens can be expressed as % of
total lymphocytes evaluated or numbers of CD16+ CD56+ and CD69+ in the
PBMC population per μl of whole blood.

[0206] Similarly, the data in FIG. 2 show that the same results are
achieved if patients are selected on the basis of < or >35 CD16+
CD56+ CD69+ lymphocytes per μl of whole blood (based on measurement
using PBMC). Patients with <35 CD16+ CD56+ CD69+ lymphocytes per μl
of whole blood (based on measurement of PBMC) survive longer (median
survival=19.9 months) than patients with ≧35 CD16+ CD56+ CD69+
lymphocytes per μl of whole blood (based on measurement of PBMC)
(median survival=9.9 months).

[0207] The data in FIGS. 3 and 4 demonstrate that the effect of selecting
patients based on the expression of CD16, CD56 and CD69 on their
lymphocytes is restricted to patients receiving the vaccine. FIG. 3 shows
that patients with < or >3.45% CD16+ CD56+ CD69+ lymphocytes at
baseline have the same survival expectancy. FIG. 4 shows that the
observation using 35 CD16+ CD56+ CD69+ lymphocytes per μl whole blood
at baseline.